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Current Biology 23, 323–327, February 18, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.01.010
Report
Ant Pupae Employ Acoustics
to Communicate Social Status
in Their Colony’s Hierarchy
Luca P. Casacci,
1
Jeremy A. Thomas,
2
Marco Sala,
1
David Treanor,
2
Simona Bonelli,
1
Emilio Balletto,
1
and Karsten Scho
¨nrogge
3,
*
1
Department of Life Sciences and Systems Biology,
University of Turin, Via Accademia Albertina, 10123 Turin, Italy
2
Department of Zoology, University of Oxford,
South Parks Road, Oxford OX1 3PS, UK
3
CEH Wallingford, NERC Centre for Ecology & Hydrology,
Maclean Building, Benson Lane, Wallingford OX10 8BB, UK
Summary
The possession of an efficient communication system and
an ability to distinguish between young stages are essential
attributes that enable eusocial insects to live in complex
integrated societies [1–4]. Although ants communicate
primarily via chemicals, it is increasingly clear that acous-
tical signals also convey important information, including
status, between adults in many species [5–9]. However, all
immature stages were believed to be mute [7]. We confirm
that larvae and recently formed pupae of Myrmica ants are
mute, yet once they are sclerotized, the pupae possess a fully
functioning stridulatory organ. The sounds generated by
worker pupae were similar to those of workers but were
emitted as single pulses rather than in the long sequences
characteristic of adults; both induced the same range and
intensity of benevolent behaviors when played back to
unstressed workers. Both white and sclerotized pupae
have a higher social status than larvae within Myrmica colo-
nies, but the latter’s status fell significantly after they were
made mute. Our results suggest that acoustical signals
supplant semiochemicals as a means of identification in
sclerotized pupae, perhaps because their hardened integu-
ments block the secretion of brood pheromones or because
their developing adult secretions initially differ from overall
colony odors [5, 10].
Results and Discussion
Pupal Sound Production
The main means of recognition and communication that permit
up to a million individuals in an ant society to function as
a single ‘‘superorganism’’ is by chemical cues, often modu-
lated by tactile stimuli [1, 2, 4]. Members of the same society
typically share a cocktail of hydrocarbons that provides a
distinctive ‘‘gestalt’’ odor across the colony, allowing workers
to discriminate between kin and strangers [5, 6]. Additional
variation between individuals’ profiles permits recognition
of—and appropriate responses to—nestmates of different
sex, caste, and developmental stage [10–14]. For example,
when a colony is perturbed, the workers quickly rescue and
retrieve the brood, including dummies treated with larval ex-
tracts [15, 16]. In the well-studied Myrmicine genus Myrmica,
brood recognition by pheromones is supplemented by tactile
cues, including larval turgidity, hairiness, size, shape, and
surface properties [4]; a social hierarchy exists between the
different young stages: small larvae are killed and fed to
larger larvae in times of food shortage, and a distinct order
of rescue occurs—starting with pupae, followed by large
larvae and finally by small larvae and eggs—whenever a colony
is disturbed [17, 18].
The role of acoustic signaling has recently received in-
creased interest [7–10], but, to our knowledge, there was no
evidence that the young stages of any ant could communicate
using sound. On the contrary, previous studies indicated that
the immature stages of Myrmica were mute [7, 19], although
older, sclerotized pupae may not have been investigated.
Scanning electron microscopy revealed, however, the pres-
ence of a fully formed stridulatory organ on the developing
imago within sclerotized ant pupae, similar to that on adult
workers and queens (Figure 1). The organ consists of a
minutely ridged file (Figures 1C and 1D; pars stridens), located
on the middorsal edge of the fourth abdominal segment, and
of a spike (plectrum) projecting from the rear edge of the
postpetiole. However, compared with adults, the scope for
the pupa to play one surface rapidly against the other was
constrained due to the thin pupal cuticle that encompassed
it (Figures 1Aand 1B). Emerging stridulatory organs were
also recognizable on the soft abdomens of newly formed white
pupae but were absent from larvae.
We recorded larvae and white pupae for a total of 40 hr, but
no sounds or substrate-borne vibrations were detected. In
contrast, sclerotized (nascent worker) pupae readily produced
acoustic signals which resembled those of adult workers and,
to a lesser extent, queens in their frequency and intensity, but
which consisted of single pulses rather than the streams of
‘‘song’’ emanating from both adult castes (Figure 2A). Using
a multivariate approach over three sound parameters, the
normalized Euclidean distances (mean 6SD) within samples
of M. scabrinodis pupae, queens, and workers were respec-
tively 0.88 60.32, 0.52 60.30, and 1.00 60.59 (Figure 2A).
Principal component analysis (PCA) was also conducted on
the three sound parameters recorded from groups of 6
M. scabrinodis sclerotized pupae, 1 individual worker, and 1
queen from each of 10 M. scabrinodis nests: the first and
the second principal components accounted for 79.1% and
20.9% respectively, i.e., explaining all the variance (Figure 2B).
Nested analysis of similarity (ANOSIM) of the Euclidean
distance matrix showed a clear separation between the
signals of sclerotized pupae, workers, and queens (overall:
R= 0.778, p < 0.001; for component distances: sclerotized
pupae: distance
workers
= 2.52 61.00, ANOSIM R= 0.941, p =
0.001; distance
queens
= 3.16 60.96, ANOSIM R=1,p=
0.001). As expected, the signals emitted by sclerotized
(nascent worker) pupae were significantly closer to the stridu-
lations of workers than to those of queens (two-sample t test:
t = 10.198, df = 198, p < 0.001).
The adults of many ant species stridulate to nestmates
[20, 21], although acoustical communication by their immature
stages has not been previously described. Because the
active organ formed part of a nascent adult developing inside
the sclerotized M. scabrinodis pupa, we might expect to find
*Correspondence: ksc@ceh.ac.uk
similar acoustical communications, after the first few days
of pupal lives, among the four subfamilies of ants which
also possess a stridulatory organ, namely the Ponerinae,
Nothomyrmecinae, Pseudomyrmecinae, Myrmicinae.
Worker Ant Responses to Pupal Sounds
The responses of otherwise undisturbed M. scabrinodis
workers to recordings of the sounds emitted by their sclero-
tized pupae were compared with playbacks of their own
(worker) recordings and of white noise in three randomly as-
signed containers, simultaneously replicated twenty times.
No antagonistic or alarmed ant behavior occurred during
playback experiments, but five benevolent responses were
observed, the first two involving attraction and the rest involv-
ing reactions: (1) walking—the worker was attracted to the
speaker but walked over it without stopping on it; (2) alerting—
the worker abruptly changed direction to pass onto the
speaker; (3) antennating—the worker antennated the speaker
for at least 3 s; (4) guarding—the workers rested in an alert
on-guard poise (sensu; [7]) on the speaker for at least 5 s; (5)
digging—the worker dug into the soil surrounding the speaker.
Linear mixed-effect models showed that worker reactions to
the three sound stimuli were significantly different for all
observed behaviors except digging, which, however, was
never elicited by white noise (Figure 3). Thus, compared with
white noise, both pupal and worker sounds always induced
significantly more instances of walking, alerting, antennating,
and guarding by Myrmica worker ants, with values of p ranging
from 0.019 to <0.0001. Yet despite the fact that pupal calls
consisted of single pulses, whereas worker stridulations
were broadcast in streams, no significant difference was found
in worker responses to these two sound stimuli (Figure 3).
The results are consistent with other observations within the
genus Myrmica that stridulations are caste specific rather than
species specific [8], and, unsurprisingly, the structure of the
stridulation organ we found in M. scabrinodis worker pupae
was identical to that of eclosed adult workers (Figures 1 and
2). Similarly, we predict that the stridulatory organ of a gyne
pupa will produce sounds similar to an adult queen and will
induce similar royal treatment from nurse workers [7]. The
constraint of an enveloping integument may explain why the
pupal sounds occurred in single pulses rather than the
complex repetitions that characterize an adult ant’s diagnostic
patterns. The fact that both types of adult and pupal stridula-
tions triggered the same intensity and range of benevolent
responses suggests that the frequency at which pulses of
sounds occur is not important for conveying information. It is
worth noting, however, that our test environment was simple
and constant, and that in nature adult ants are capable of
both producing different sounds [22] and reacting in different
ways to the same acoustics [23], depending on the context
in which the signal is transmitted or received. Furthermore,
our acoustics were tested in isolation, whereas in nature they
may be modulated by chemical or tactile cues, and vice versa
[1, 2]. Thus, we suspect that tended pupae in natural colonies
may possess a wider acoustical repertoire than observed here
and that worker responses to them may be more complex.
Social Status of Normal and Mute Myrmica Pupae
As has been reported for other Myrmica species [17, 18], we
found that M. scabrinodis workers rescued living pupae, as
Figure 1. The Stridulatory Organ of Sclerotized Pupae of Myrmica
scabrinodis
(A and B) Location of the acoustical organ (arrow) beneath the integument of
an intact pupa.
(C) Pupa with integument removed.
(D) Pars stridens on pupa with integument removed.
Figure 2. Comparison of the Acoustics of Queen,
Worker, and Sclerotized Pupae of Myrmica
scabrinodis
(A) Oscillogram, spectrogram, and single pulse
parameters.
(B) Combined effect of the three sound parame-
ters (pulse length, frequency, and intensity)
shown as the first and second component plot
of a principal components analysis over all indi-
vidual pulse measurements.
Current Biology Vol 23 No 4
324
a class (i.e., brown + white), significantly more quickly than
their larvae (Wilcoxon Mann-Whitney, Z= 6.822, p = 0.009)
after their nest was disturbed (Figure 4). However, within
these assays, using normal (i.e., nonmuted) brood items, the
white pupae were rescued ahead of both sclerotized pupae
(Z= 2.118, p = 0.026) and larvae (Z=23.177, p < 0.001), with
no significant difference being found between sclerotized
pupae and larvae (Z=21.399, p = 0.168), although the latter
were, on average, rescued last (Figure 4).
The pattern of rescue changed with recently killed brood:
i.e., brood still coated with its full cocktail of recognition
pheromones [24, 25] but which was mute and immobilized
(Figure 4). The mute sclerotized pupae were the last to be
rescued, significantly behind white pupae (Z= 3.326, p <
0.001) and larvae (Z= 2.306, p = 0.021). White pupae were on
average rescued first, but not significantly ahead of larvae
(Z= 1.5875, p = 0.107). Wilcoxon signed rank tests were also
used to directly compare the shift in order in each brood
type between the normal and mute trials: sclerotized pupae
shifted to being rescued significantly after the other brood in
the mute trials (Z=224.500, df = 10, p = 0.0098), but there
was no significant shift in the order of recovery of white pupae
or larvae between the two experiments (Z=24.500, df = 10,
p = 0.6719 and Z=215.500, df = 10, p = 0.1309, respectively).
It was impractical to record the acoustics of Myrmica pupae
during the rescue experiment, but the shift in rank for the
brown pupae that could and could not stridulate indicates
that this is linked to the stridulations. Of course, dead brood
cannot move, either; e.g., larvae cannot beg, but the lack of
any significant difference in the relative order of rescue of
white pupae and larvae during the mute assays compared
with the living trials supports previous conclusions [24, 25]
that the chemical and tactile signals involved in brood recog-
nition are not compromised by this treatment.
The preference afforded to living white pupae after colony
perturbation was unexpected. We predicted that the calls of
sclerotized pupae would attract preferential worker attention,
perhaps explaining why pupae as a group were selected
ahead of larvae or eggs in previous ant rescue experiments
[17, 18]. A possible explanation is that, rather than elevating
the social level of sclerotized pupae through the possession
of an additional cue, their acoustics may replace brood-recog-
nition pheromones, perhaps because the hardened integu-
ment blocks the secretions from their own glands or reduces
their ability to absorb colony odors. An alternative explanation
is that hydrocarbons secreted by the developing imago within
a sclerotized pupa not only replace or overscore the phero-
mones of brood with soft cuticles, but—like the secretions of
the callow adults that they will shortly become—differ some-
what from the overall gestalt odor of their colony, making
them less recognizable as nestmates using chemical cues
alone.
General Conclusions
Our results support a growing body of work—facilitated by the
increased sophistication of affordable sound equipment—that
suggests that acoustical communication plays a greater and
more varied role in influencing ant social behavior than was
previously thought (e.g., see [7, 8, 20–22, 26]).
The recognition, not only of brood in ant societies but also of
different types of brood, including nestmate and nonnestmate
brood, has received much recent attention. Although it seems
clear that chemical, tactile, behavioral, and now acoustic cues
can be important in brood recognition [4, 10, 25], the precise
role of each cue is still poorly understood. For instance, on
current evidence we have suggested that acoustical signals
are caste specific but not species (let alone kin) specific.
On the other hand, the cuticular hydrocarbon signatures
described on brood are often impoverished and dominated
by saturated alkanes that are not thought to convey informa-
tion [27, 28]. If this were the case, brood would be chemically
transparent [10, 29] and distinctive to workers only if other
Figure 3. Responses of Myrmica scabrinodis Workers to Broadcasts of
Worker and Pupal Acoustics and White Noise
Five benevolent but no antagonistic behaviors were observed: the same
letter indicates no significant difference within each type of behavior;
different letters indicate a significantly different response. Compared with
white noise, linear mixed-effect model likelihood ratios are (1) walking
LR
pupa
= 11.082, df = 4, p = 0.001; LR
worker
= 8.097, df = 4, p = 0.004; (2)
alerting LR
pupa
= 23.232, df = 4, p < 0.0001; LR
worker
= 20.518, df = 4,
p < 0.0001; (3) antennating LR
pupa
= 8.425, df = 4, p = 0.004; LR
worker
=
17.154, df = 4, p < 0.0001; and (4) guarding LR
pupa
= 5.476, df = 4, p =
0.019; LR
worker
= 11.419, df = 4, p = 0.001. Likelihood ratios comparing pupal
and worker acoustics are (5) walking LR = 0.296, df = 4, p = 0.587; (6) alerting
LR = 0.145, df = 4, p = 0.704; (7) antennating LR = 2.278, df = 4, p = 0.131; and
(8) guarding LR = 1.441, df = 4, p = 0.230.
Figure 4. The Hierarchical Status of Myrmica Brood Items
Box plots illustrate the order in which worker ants rescued sclerotized
(brown) pupae, young white pupae, and larvae after their nest was perturbed
by exposure to light: vertical line = median rank of rescue, box = 25
th
–75
th
percentiles, whiskers = one standard deviation below and above the
mean of the data. White boxes show ‘‘normal’’ live ant brood (overall
Kruskal-Wallis H
n
= 11.182, df = 2, p = 0.003), and gray boxes show results
for ‘‘mute’’ recently dead brood (H= 26.347, df = 2, p < 0.001).
Acoustic Signaling of Ant Brood Status
325
undescribed brood pheromones exist [30]. Recent studies,
however, have shown that nonnestmate brood is often
adopted into an ant society as a quick and efficient way of
increasing the workforce, whereas behavioral experiments
show that any kin brood is always chosen first, indicating a
clear ability among workers to recognize immature nestmates
within their species [10, 31].
Notwithstanding the predominant use of semiochemicals in
ant communications, many species generate acoustical
signals through a stridulatory organ or by drumming their
gaster. Once considered a weak form of communication,
restricted to spreading alarm or modulating responses to
other signals [1, 32–34], it is increasingly clear that acoustics
is used to convey a greater variety of information between
nestmates as well as to signal an individual’s social status
[7, 8]. We suspect that acoustics may be a more flexible means
of signaling and conveying information between both adult
and immature ants than is generally recognized [22].
Experimental Procedures
Field Collection and Culture
Myrmica scabrinodis nests (n = 10) were collected in July 2011 at Walling-
ford (UK), set as standardized laboratory ant colonies with >100 workers
in 12.5 cm 38cm32 cm Perspex containers, and maintained on a diet
of sugar and Drosophila larvae [35]. All colonies contained a minimum of
ten larvae, ten white pupae, and ten sclerotized pupae.
Scanning Electron Microscopy
We used dissection and scanning electron microscopy to investigate the
presence of stridulatory organs on ant brood. Two M. scabrinodis larvae
and two white and two sclerotized pupae from two ant colonies were kept
in 70% ethanol, and one item per category was dissected between the post-
petiole and the abdomen to expose the pars stridens and the plectrum. The
whole individuals and the two ant parts were mounted on the same steel
stub and coated with gold, and the samples were scanned using a Cam-
bridge Stereoscan S360 scanning electron microscope. M. scabrinodis
white pupae and larvae were dried in hexamethyldisilazane to avoid cell
structure disruption before coating. The SEM operated at 20–25 kV.
Sound Recordings
We recorded sounds of clusters of six M. scabrinodis larvae and six white
and six sclerotized pupae from ten M. scabrinodis nests. Separate record-
ings were made of individual queens and workers taken from the same test
colonies. The recording equipment consisted of a 12.5 cm 38cm32cm
recording chamber with a moving-coil miniature microphone attached
through the center. A second microphone of the same type was used to
record ambient noise but in antiphase. An amplifier was attached to each
microphone and calibrated to maximize the noise cancellation of ambient
noise from the two microphones, leaving the signal from the recording
chamber. The resulting signal was processed through two-stage low-noise
amplification before being digitally recorded on a laptop computer, using
Audacity 1.3 Beta (http://audacity.sourceforge.net/). To further reduce
ambient noise and interference, the equipment was powered by a 12 V gel
cell battery, and the recording chamber and microphones were placed
inside an anechoic chamber. Sounds were recorded for 20 min periods
starting 10 min after items were introduced into the recording chamber.
Recordings were sampled at 44.10 kHz and 32-bit resolution. Frequency
information was obtained through fast Fourier transformation (FFT; width
1,024 points). Spectrograms were obtained at Hanning window function
with 512 bands resolution. We selected 20 good quality pulses from each
track and measured dominant frequency (Hz), pulse length, and sound
amplitude (dB) using Audacity 1.3 Beta. Based on the three sound parame-
ters, single pulses were ordinated by principal components analysis (PCA).
To test whether sound differed between groups, we calculated the pairwise
normalized Euclidean distance over all three parameters and used a nested
(‘‘colony’’ within ‘‘group’’) ANOSIM implemented in Primer v6 (Primer-E
Ltd.). The sound parameters were log(x+1) transformed. We calculated
the average pairwise distances and used a two-sample t test to compare
differences between group distances.
Worker Ant Responses to Sound Recordings
Behavioral assays were carried out in three 7 cm 37cm35 cm Perspex
arenas with the speaker attached at the bottom of the box and sealed on
the outside with Blu-Tack. The speaker was covered with a thin layer of
slightly wet soil. Ten workers from the same M. scabrinodis colony were
placed in each arena and allowed to settle for 10 min before being played
one of the three test sounds (M. scabrinodis worker, sclerotized pupae
sound, and white noise). The sounds were produced by MP3 players playing
loops of the original recordings, with each volume adjusted to the natural
level by attaching the speaker to the microphone of the recording equip-
ment and by calibrating to the same levels reached during recording.
Each trial lasted 30 min: counts were made of all instances of antagonistic
or attractive behaviors, during periods of one minute for each box, and in
sequence between the three treatments, i.e., S10 min for each sound per
trial. Each playback experiment was repeated 20 times, using fresh ants
from ten different M. scabrinodis colonies (i.e., twice for each colony). The
source of sound for each arena was randomly assigned before each trial
was replicated to control for possible positional effects. Between each trial,
new soil was introduced and all the equipment, including speakers and
arenas, was cleaned with absolute alcohol and rinsed with distilled water.
The effect of sound stimulus on the five worker ant behaviors was analyzed
in a linear mixed-effects model with ‘‘colonies’’ as a random factor using the
software R-2.15.0 [36].
Experiment to Measure the Order in which Workers Rescued Different
Brood Items
The arena used for the brood-rescue experiment consisted of two adja-
cent chambers of 7 32 cm communicating at one end. We placed eight
Myrmica larvae, eight white pupae, eight sclerotized pupae, and ten
workers on a 0.4 cm
3
moist sponge (to maintain humidity) at the end of
one chamber, which was then covered with a transparent glass. The other
chamber was covered with a dark glass. After 10 min of resting in the dark,
we shone a 60 W light placed 10 cm away onto the chamber containing the
worker ants and brood, to create a high level of stress which induced
workers to rescue the exposed brood and carry it into the dark chamber.
The order in which each item of brood was rescued was recorded. The
experiment was then repeated after placing all brood items from a colony
in a freezer (220C) for 20 min, thus killing the brood to make them mute
(and immobile). Brood items were then left at room temperature for 5 min
to return to normal temperature. Immediately after this period, the same
procedures as before were used to make rescue experiments. Previous
studies [24, 25] have established that in assays conducted only a short
time after immature ants are killed, the chemicals responsible for brood
recognition remain present in approximately the same quantities as in
the live brood.
Statistical analyses were performed using the package ‘‘coin’’ provided
with the software R-2.15.0 [36, 37]. Kruskal-Wallis tests were used to
compare the rescue orders of different brood categories between nonmute
and mute treatments. Subsequent pairwise comparisons of median
rescue order between brood categories within the same treatment
were made using Wilcoxon Mann-Whitney tests; p values were calculated
against a null distribution generated from data using a Monte Carlo
resampling. Direct comparisons of the same brood categories between
normal and mute treatments were made using paired Wilcoxon signed
rank tests.
Acknowledgments
Research was funded within the project CLIMIT (Climit Change Impacts on
Insects and their Mitigation; Settele and Ku¨ hn [2009] [38], Thomas et al.
[2009] [39]), funded by Deutsches Zentrum fu¨ r Luft-und Raumfahrt-Bundes-
ministerium fu¨r Bildung und Forschung (Germany); Natural Environment
Research Council (NERC) and Department for Environment, Food, and Rural
Affairs (UK); Agence nationale de la recherche (France); Formas (Sweden);
and Swedish Environmental Protection Agency (Sweden) through the
FP6 BiodivERsA Eranet. Part of the research was funded by the Italian
Ministry of Education, University, and Research (MIUR) within the project
‘‘A multitaxa approach to study the impact of climate change on the biodi-
versity of Italian ecosystems.’’
Received: December 3, 2012
Revised: January 2, 2013
Accepted: January 2, 2013
Published: February 7, 2013
Current Biology Vol 23 No 4
326
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